Cells were seeded in a black wall, clear-bottom 96-well plate at a concentration of 10,000 cells/well, in 200 μL culture medium. After 24 h of incubation at the previously described conditions, cells were washed with PBS and fixed with 4% PFA at room temperature for 20 min. Next, one set of the samples was permeabilized with 0.5% Triton X Buffer. Anti-GPER1 (Biorbyt, St Louis, MO, USA, Catalogue No.: orb10740), anti-ERα (Thermo Fisher Scientific, Catalogue No.: PA1-308) and anti-Erβ (Thermo Fisher Scientific, Catalogue No.: MA524807) primary antibodies were added overnight. Secondary antibodies (ATTO 647N, Sigma Aldrich, Merck KgaA, Darmstadt, Germany, Catalogue No.: 40839, and Alexa Fluor 488, Thermo Fisher Scientific, Catalogue No.: A32731) was added 18 h later. Nucleus staining was conducted with Hoechst 33342 (Thermo Fisher Scientific, Catalogue No.: H3570). Secondary antibody without the primary was used as control. The samples were imaged by Celldiscoverer 7 system using 50 × Plan-Apochromat λ/0.35 NA water immersion objective with 2 × tube lens (Carl Zeiss AG, Jena, Germany) using the Z-stack function (minimum 20 Z-stacks per image). Then, all images were deconvolved to enhance the signal-to-noise ratio using ZEN Blue 2.6 software (Carl Zeiss AG, Jena, Germany). The intensity values and the number of nuclei were determined by ImageJ software (NIH, Bethesda, MD, USA).
Anti erα
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-ERα is a laboratory reagent used for the detection and quantification of the Estrogen Receptor alpha (ERα) protein. It is a specific antibody that binds to the ERα protein, allowing researchers to study its expression and distribution in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Atto 647n, by Merck Group (1 mentions)
Anti erβ, by Thermo Fisher Scientific (1 mentions)
Zen blue 2, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Celldiscoverer 7 system, by Zeiss (1 mentions)
Neomarker, by Lab Vision (1 mentions)
Anti cdk1, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Odyssey system, by LI COR (1 mentions)
Anti rabbit or anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti dnmt1, by Merck Group (1 mentions)
Anti ar, by Thermo Fisher Scientific (1 mentions)
Anti c myc, by Santa Cruz Biotechnology (1 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Anti p pi3k, by Affinity Biosciences (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg ab150077, by Abcam (1 mentions)
Anti pten, by Bioss Antibodies (1 mentions)
7 protocols using anti erα
1
Immunofluorescence Microscopy of GPER1, ERα, and ERβ
2
Immunohistochemical Analysis of Ki-67, Caspase-3, and ER-α
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Immunohistochemical staining was performed for Ki-67, activated caspase-3 and ER-α. Sections were treated with xylene to remove paraffin and rehydrated through a series of alcohols, washed with phosphate-buffered saline (PBS) and then microwaved in citrate buffer (pH 6) for antigen retrieval. Non-specific binding was blocked by incubation with normal goat serum for 1 h. The sections were incubated overnight at 4°C in a humid chamber with a monoclonal antibody against Ki-67 and activated caspase-3 in a dilution of 1:100 (NeoMarkers; Lab Vision, Fremont CA, USA) and polyclonal anti-ER-α (Thermo Fisher Scientific, Waltham, MA, USA) in a dilution of 1:400 using the streptavidin-peroxidase method according to the manufacturer's protocol. Corresponding biotinylated secondary antibody was applied to the sections for 1 h in a humidified chamber at room temperature. Slides were rinsed with PBS, and the sections were then counterstained with Mayer's haematoxylin. Positive controls for Ki-67 were from the normal tonsil, for caspase-3 from the placenta and for ER-α from the breast carcinomas. Negative control was made by the exclusion of the primary antibody (Kobayashi et al. 2013) .
3
Western Blot Analysis of Cell Signaling
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Whole cell lysates were prepared from cells and subjected to western blot analyses as previously described (26 (link)). Western blots were incubated with primary antibodies from Cell Signaling Technology [anti-CDK1 (catalog #77055), anti-ERα (anti-AR, anti-PSA (catalog #5365), and anti-c-Myc (catalog #9402)], from Santa Cruz Biotechnology (Dallas, TX, USA) [anti-GSK3 (catalog #sc-7291), anti-AR (catalog #sc-7305), and anti-ERα (catalog #sc-543)], from Invitrogen [anti-DNMT1 (catalog #PA5-80557)], from Millipore [phosphoserine(catalog #AB1603)] and from Sigma-Aldrich [anti-GAPDH (catalog #MAB374)]. Anti-pDNMT1 (S714) is an in-house antibody. Blots were then incubated with anti-rabbit or anti-mouse secondary antibody (catalog #31460 and 31430, respectively, Thermo Scientific, Waltham, MA, USA) at room temperature. Signals were detected using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific, catalog #34094), visualized using the LI-COR Odyssey System (LI-COR Biosciences, Lincoln, NE, USA), and quantified using the ImageJ software.
4
Quantifying Cellular Signaling Pathways
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CMT-U27 and CF41.Mg cells were cultured on gelatin-coated coverslips. Cells were fixed with 2% paraformaldehyde for 20 min at 4 °C and permeabilization using 0.5% Triton X-100 in PBS for 10 min. Blocking was performed using 5% animal serum (donkey or goat) in 2% bovine serum albumin in PBS for 1 h at room temperature. Subsequently, the cells were treated with primary antibodies against anti-ERβ (1:200; Abcam), anti-ERα (1:200; Invitrogen), anti-p-PI3K (1:200; Affinity Biosciences), and anti-PTEN (1:200; Bioss) in a blocking solution at 4 °C overnight. The cells were incubated with Alexa FluorTM 594 conjugated goat anti-mouse IgG (A-11005; 1:1000; Invitrogen) or Alexa Fluor® 488 conjugated goat anti-rabbit IgG (ab150077; 1:1000; Abcam). The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The cells were then mounted using mounting medium (Dako, Carpinteria, CA, USA) and images were captured using a THUNDER Imager 3D Live Cell & 3D Cell Culture System (Leika Microsystems, Wetzlar, Germany). The mean fluorescence intensity for each channel in the three distinct regions was quantified using ImageJ software (version 1.52a). The fluorescence intensity was expressed relative to that of the control.
5
Western Blot Analysis of Reproductive Signaling
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The tissue or cell samples were homogenized in RIPA buffer containing protease inhibitor cocktail. Protein per sample were loaded and separated in a 10% SDS-PAGE gel. The separated samples were transferred to nitrocellulose membranes. After blocking with 5% nonfat milk, the membranes were incubated to the primary antibodies anti-FSHR (1:500; Abcam, ab75200), anti-PKA (1:1000; CST, #4782), anti-Kir6.2 (1:1000; Santa Cruz, sc390104), anti-pKir6.2 (1:1000; Invitrogen, PA5-40157), and anti-CaV1.2 (1:1000; Abcam, ab270987), anti-pCaV1.2 (1:1000; Invitrogen, PA5-64748), anti-ERα (1:1000; Invitrogen, MA1 − 80216), anti-ERβ (1:1000; Invitrogen, PA1 − 311), anti-GPCR30 (1:1000; Abcam, ab260033), anti-β-Actin (1:10,000; Proteintech, 66009-1-lg) or anti-GAPDH (1:10,000; Proteintech, 60004-1-lg) at 4 °C overnight. Following incubation with the corresponding secondary antibodies, images were taken by BIO-RAD ChemiDoc MP and analyzed by Image J 1.52a software.
6
Western Blot Analysis of Protein Expression
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Cells were rinsed once with ice-cold PBS then scraped from plates using SDS lysis buffer (Beyotime, #P0013G) containing protease inhibitors (Beyotime, #P1005), and lytic at 100 °C for 10 min. Then, centrifugation at 12,000 × g for 10 min at 4 °C to collect the supernatant, and measurement the protein concentrations by BCA Protein Assay Kit (Beyotime, #P0012S). Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, IPVH0010), after blocking with 5% w/v nonfat milk at RT for 1 h, the membranes were incubated with specific primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies at RT for 1 h. Blots were visualized by chemiluminescence reagent (Millipore, #WBLUR0500). The band density was quantified using Quantity One Software (v4.62, Bio-Rad). The primary antibodies included anti-ERα (Invitrogen, #MA1-12692, 1:200), anti-CUL4B (Proteintech, #12916-1-AP, 1:1000), anti-Ubiquitin (CST, #43124, 1:1000), anti-FasL (abcam, #ab15285, 1:1000), anti-PARP (CST, #9532, 1:1000), anti-GAPDH (CST, #5174, 1:1000), anti-Caspase3 (CST, #9662, 1:1000), Anti-mouse IgG, HRP-linked Antibody (CST, #7076, 1:5000) and Anti-rabbit IgG, HRP-linked Antibody (CST, #7074, 1:6000). The complete unedited blots are shown in Source data.
7
Western Blot Analysis of Statin and E2 Effects
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